RESUMO
Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with â¼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of â¼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was â¼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
Assuntos
DNA Girase/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Catálise , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
OBJECTIVE: To establish a method for diagnosis of freshwater drowning by amplifying gyrB and 16S rRNA genes of Aeromonas hydrophila using PCR coupled with capillary electrophoresis (CE). METHODS: DNA samples were extracted from human, 18 planktons (including Candida albicans, Aeromonas hydrophila, and 16 species of algae), and 30 cases of tissue samples (including the lung, liver, and kidney, all examined with microwave digestion-vacuum filtration-automated scanning electron microscopy) from human cadavers, including 28 freshwater drowning victims and 2 with natural death. The DNA samples were amplified with the primer AH (for gyrB gene) and primer Ah (for 16S rRNA gene), and the products were analyzed with CE. RESULTS: PCR amplification followed by CE yielded negative results for DNA of human, Candida albicans and 16 species of algae, whereas a positive result was found for Aeromonas hydrophila DNA with PCR products of 195 bp (with primer AH) and 350 bp (with primer Ah). In the 28 drowning cases, the detection rates of Aeromonas hydrophila using primer AH were 96.4% in the lung tissue, 71.4% in the liver tissue, and 60.7% in the kidney, as compared with the rates of 75.0%, 42.9%, and 32.1% using primer Ah, respectively. The positive rates for Aeromonas hydrophila in the organs of the drowning victims were 82.1% and 53.6% with primer AH and primer Ah, respectively. The detection showed negative results in the 2 cases of natural deaths. The two primers produced significantly different detection rates of Aeromonas hydrophila (P<0.05). CONCLUSION: PCR coupled with CE for detecting gyrB gene of Aeromonas hydrophila has a high sensitivity in assisting a diagnosis of freshwater drowning. Detection of both the gyrB gene and 16S rRNA gene of Aeromonas hydrophila can yield more convincing evidence of the diagnosis of freshwater drowning.
Assuntos
Aeromonas hydrophila/genética , Afogamento/diagnóstico , RNA Ribossômico 16S/isolamento & purificação , Cadáver , DNA Girase/isolamento & purificação , Primers do DNA , DNA Bacteriano/isolamento & purificação , Afogamento/microbiologia , Eletroforese Capilar , Humanos , Rim , Fígado , Pulmão , Reação em Cadeia da PolimeraseRESUMO
The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.
Assuntos
Antibacterianos/farmacologia , Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/enzimologia , Cloroplastos/efeitos dos fármacos , Ciprofloxacina/farmacologia , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Substituição de Aminoácidos , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Cloroplastos/ultraestrutura , DNA Girase/química , DNA Girase/genética , DNA Girase/isolamento & purificação , Resistência a Medicamentos , Técnicas de Inativação de Genes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Forma das Organelas/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/ultraestrutura , Mutação Puntual , Conformação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMO
A number of important protozoan parasites including those responsible for toxoplasmosis and malaria belong to the phylum Apicomplexa and are characterised by their possession of a relict plastid, the apicoplast. Being required for survival, apicoplasts are potentially useful drug targets and their attractiveness is increased by the fact that they contain "bacterial" gyrase, a well-established antibacterial drug target. We have cloned and purified the gyrase proteins from the apicoplast of Toxoplasma gondii (the cause of toxoplasmosis), reconstituted the functional enzyme and succeeded in characterising it. We discovered that the enzyme is inhibited by known gyrase inhibitors and that, as well as the expected supercoiling activity, it is also able to decatenate DNA with high efficiency. This unusual dual functionality may be related to the apparent lack of topoisomerase IV in the apicoplast.
Assuntos
DNA Girase/metabolismo , Toxoplasma/metabolismo , Adenosina Trifosfatases/metabolismo , Cálcio/metabolismo , DNA/metabolismo , DNA Girase/química , DNA Girase/isolamento & purificação , DNA Topoisomerases/química , DNA Topoisomerases/isolamento & purificação , DNA Topoisomerases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Holoenzimas , Concentração Inibidora 50 , Multimerização Proteica , Inibidores da Topoisomerase II/farmacologiaRESUMO
DNA gyrase is a type IIA topoisomerase found in bacteria but not in humans. The enzyme is required for bacterial DNA replication and transcription, and is an important antibacterial target that is sensitive to the widely-used fluoroquinolone drugs. Due to the emergence of fluoroquinolone resistance, the discovery of new classes of drugs that target DNA gyrase is urgent. The DNA gyrase holoenzyme is a heterodimer of subunit pairs (A2B2). The 90 kDa A subunits bind, cleave, and rejoin double stranded DNA. The enzyme introduces negative supercoils into closed circular bacterial DNA using ATP hydrolysis catalysed by the 70 kDa B subunits. Subdomains of DNA gyrase subunits have been crystallised for structural analysis and the resulting models used to improve drugs that target the DNA binding region and active site. While crystal structures are available for topoisomerase IV complexes with cleaved DNA, there is none for the complete DNA gyrase complex with substrate DNA bound. Thermophiles offer significant advantages in obtaining stable enzymes for structural and functional studies. In order to develop a capability for drug screening and structure-directed drug discovery we have reconstituted a functional and drug-sensitive DNA gyrase complex using heterologously expressed subunits from the thermophile Thermus thermophilus.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Girase/química , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Estabilidade Enzimática , Hidrólise , Modelos Moleculares , Thermus thermophilus/química , Thermus thermophilus/genéticaRESUMO
A series of fourteen (A1-A14) new qunioline based chalcones were synthesized by condensing 2,7-dichloro-8-methyl-3-formyl quinoline with acetophenone and acetylthiophenes, and subsequently characterized by IR, NMR and Mass spectroscopy. All the compounds were screened for antibacterial activities and found potentially active antibacterial agents. Bioassay, theoretical and dockings studies with DNA gyrase (the enzyme required for super coiling of DNA of bacteria) results showed that the type and positions of the substituents seemed to be critical for their antibacterial activities. The bromo and chloro substituted chalcone displayed high anti-bacterial activity. The A4 and A6 showed high interaction with DNA gyrase, contributing high free binding energy (ΔG -8.18 and -8.88 kcal).
Assuntos
Antibacterianos/farmacologia , Chalconas/farmacologia , DNA Girase/metabolismo , Quinolinas/química , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Chalconas/síntese química , Chalconas/química , DNA Girase/química , DNA Girase/isolamento & purificação , Relação Dose-Resposta a Droga , Enterobacter aerogenes/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Salmonella typhimurium/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/químicaRESUMO
Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , DNA Girase/química , Mycobacterium tuberculosis/enzimologia , Adenosina Trifosfatases/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , DNA Girase/isolamento & purificaçãoRESUMO
Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.
Assuntos
Humanos , /análise , /isolamento & purificação , Ácido Nalidíxico/isolamento & purificação , Sequência de Bases , DNA Girase/isolamento & purificação , DNA Topoisomerases/análise , DNA Topoisomerases/isolamento & purificação , Predisposição Genética para Doença , Mutação , Métodos , Pacientes , MétodosRESUMO
Fluoroquinolone (FQ) resistance in Pseudomonas aeruginosa has spread. The purpose of this study was to investigate the correlation between representative FQ, i.e. levofloxacin (LVX), resistance and mutations in the gyrA and parC genes of P. aeruginosa clinical isolates from the urine of urinary tract infection patients and their rapid detection by denaturing high-performance liquid chromatography (DHPLC). The susceptibility to LVX of 114 clinical isolates was measured and the quinolone resistance-determining regions (QRDRs) in the gyrA and parC genes of these isolates were sequenced. DHPLC was undertaken to correlate the distinctive chromatograms with their DNA mutation patterns. Among 114 isolates tested, 22 isolates (19.3%) were resistant to LVX. Six amino acid mutations were detected (Thr83Ile, Asp87Tyr and Asp87Asn in gyrA and Ser87Leu, Ser87Trp and Glu91Arg in parC), existing alone or in combination. There were 10 kinds of mutation patterns. The presence of two or more kinds of mutation significantly correlated with LVX resistance compared with the wild-type or a single mutation (P<0.0001). DHPLC data identified the number of amino acid mutations with reproducibility distinguishable by peak number and profile of the DHPLC chromatogram. In conclusion, two or more mutations in gyrA and parC were significantly related to LVX resistance in P. aeruginosa. DHPLC facilitated the detection of resistant alleles, providing a rapid (5 min per sample), economical (96 samples per run) and reliable technique for characterising LVX resistance in P. aeruginosa. This rapid detection system could forecast LVX resistance by the DHPLC profile.
Assuntos
DNA Girase/genética , DNA Topoisomerase IV/genética , Fluoroquinolonas/farmacologia , Mutação de Sentido Incorreto , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Urinárias/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , DNA Girase/isolamento & purificação , DNA Topoisomerase IV/isolamento & purificação , Farmacorresistência Bacteriana , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Adulto JovemRESUMO
Antimicrobial treatment of animals may select resistance in Campylobacter to antimicrobial agents belonging to several classes of compounds. We investigated the effect of widely used aminopenicillin therapy on the minimum inhibitory concentration (MIC) levels in porcine Campylobacter coli isolates and investigated the presence of a ß-lactamase gene and ß-lactamase production. Epidemiological cut-off values (ECOFFs) were applied to detect decreased susceptibility. Fifty-three isolates were obtained from aminopenicillin-treated (ampicillin or amoxicillin) sows and piglets during and up to 3 weeks post-treatment. All isolates had ampicillin MICs below the ECOFF (≤ 8 µg/mL). An additional 63 isolates were sampled before treatment or from other untreated sows and piglets. Of these isolates, four had ampicillin MICs above the ECOFF. All ciprofloxacin MICs were below the ECOFF (≤ 1 µg/mL), except for three isolates from untreated sows and four isolates after aminopenicillin therapy. One isolate originating from an untreated sow had an erythromycin MIC above the ECOFF (> 16 µg/mL). None of the isolates had MICs above the ECOFFs for two or three studied antimicrobials simultaneously. Of the 116 C. coli isolates, 90 (77.6%) isolates carried the bla(OXA-61) ß-lactamase gene, and 63 (70.0%) of those produced ß-lactamase. The isolates producing ß-lactamase had higher ampicillin MICs than those without the bla(OXA-61) gene and production of ß-lactamase. Proportion of the bla(OXA-61)-positive C. coli isolates was similar among untreated animals or during and after the treatment. In conclusion, C. coli isolates did not acquire high ampicillin MICs even though aminopenicillins were administered at therapeutic levels for several days. The bla(OXA-61) gene and production of ß-lactamase increased ampicillin MICs in C. coli, but the values remained mainly under the ECOFF. We also demonstrated that aminopenicillin therapy did not select simultaneously resistance to the major antimicrobials used in human therapy against campylobacteriosis (i.e., erythromycin and ciprofloxacin).
Assuntos
Amoxicilina/farmacologia , Ampicilina/farmacologia , Campylobacter coli/efeitos dos fármacos , Campylobacter coli/isolamento & purificação , beta-Lactamases/biossíntese , Animais , Antibacterianos/farmacologia , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/veterinária , Campylobacter coli/genética , Ciprofloxacina/farmacologia , DNA Girase/genética , DNA Girase/isolamento & purificação , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Finlândia , Testes de Sensibilidade Microbiana , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/isolamento & purificação , Suínos/microbiologia , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologia , beta-Lactamases/genéticaRESUMO
DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (â¼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.
Assuntos
Cromossomos Bacterianos , DNA Girase/isolamento & purificação , DNA Topoisomerase IV/isolamento & purificação , DNA Bacteriano/química , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Antibacterianos/farmacologia , Fracionamento Celular , Césio , Cloretos , Cromossomos Bacterianos/efeitos dos fármacos , DNA Girase/genética , DNA Topoisomerase IV/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Norfloxacino/farmacologia , Ligação Proteica , UltracentrifugaçãoRESUMO
Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space group P2(1)2(1)2(1), with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase.
Assuntos
DNA Girase/química , Mycobacterium tuberculosis/enzimologia , Cristalização , Cristalografia por Raios X , DNA Girase/isolamento & purificaçãoRESUMO
A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.
Assuntos
Proteínas de Bactérias/química , DNA Girase/química , Precursores Enzimáticos/química , Inteínas , Fragmentos de Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Catálise , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Girase/metabolismo , Ditiotreitol/farmacologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Precursores Enzimáticos/metabolismo , Estabilidade Enzimática , Hidroxilamina/farmacologia , Cinética , Mesna/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Processamento de Proteína/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Substâncias Redutoras/farmacologia , Synechocystis/enzimologia , Zinco/farmacologiaRESUMO
DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological form of bacterial DNA. In this study, the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus Mu50 strain was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.80â Å resolution using a synchrotron-radiation source. The crystal belonged to space group P2(1), with unit-cell parameters a = 37.28, b = 80.19, c = 50.22â Å, ß = 110.64°. The asymmetric unit contained one molecule, with a corresponding V(M) of 2.02â Å(3)â Da(-1) and a solvent content of 39.2%.
Assuntos
Proteínas de Bactérias/química , DNA Girase/química , Staphylococcus aureus/química , Cristalização , Cristalografia por Raios X/métodos , DNA Girase/isolamento & purificação , DNA Topoisomerases Tipo II/isolamento & purificação , Escherichia coli/isolamento & purificação , Estrutura Terciária de Proteína , SíncrotronsRESUMO
The present study was designed to develop a novel, rapid, direct DNA-based protocol to enumerate aeromonads in recreational waters. An Aeromonas genus-specific real-time quantitative polymerase chain reaction (Q-PCR) protocol was developed and optimized using newly designed genus-specific oligonucleotide primers derived from the gyrase B subunit (GyrB) gene. A standard curve was developed based on the PCR protocol with a minimum quantification limit of 10 cell equivalents ml(-1) achieved using an autoclaved water sample from recreational water spiked with known quantities of an Aeromonas ATCC strain. The Q-PCR protocol was validated and applied to detect and quantify the total number of aeromonads in water samples collected from two fresh water beaches on Lake Ontario. The Q-PCR protocol revealed significantly higher numbers of aeromonads in all water samples than a culture-based assay at both beaches. Foreshore sand was found to serve as a reservoir of high concentrations of Aeromonas similar to this phenomenon noted for enteric bacteria like Eschershia coli. The new real-time Q-PCR protocol facilitated the rapid quantification of total numbers of Aeromonas cells present in recreational water samples in <3 hours without culturing.
Assuntos
Aeromonas/genética , Aeromonas/isolamento & purificação , DNA Bacteriano/análise , Água Doce/análise , Água Doce/microbiologia , Reação em Cadeia da Polimerase/métodos , Praias , DNA Girase/análise , DNA Girase/isolamento & purificação , Monitoramento Ambiental/métodos , Infecções por Bactérias Gram-Negativas/prevenção & controle , Humanos , Ontário , Microbiologia da ÁguaRESUMO
OBJECTIVES: Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli. METHODS: Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes. RESULTS: Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants. CONCLUSIONS: Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.
Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Substituição de Aminoácidos , DNA Girase/genética , DNA Girase/isolamento & purificação , Análise Mutacional de DNA , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/isolamento & purificação , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Inibidores da Topoisomerase IIRESUMO
The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase. The X. albilineans gyrA and gyrB genes are not clustered with previously described albicidin biosynthesis and self-protection genes. The GyrA and GyrB products differ from Escherichia coli homologues through several insertions and through changes in several amino acid residues implicated in quinolone and coumarin resistance. Reconstituted X. albilineans DNA gyrase showed 20- to 25-fold-higher resistance than E. coli DNA gyrase to albicidin and ciprofloxacin and 8-fold-higher resistance to novobiocin in the supercoiling assay. The X. albilineans DNA gyrase is unusual in showing a high degree of distributive supercoiling and little DNA relaxation activity. X. albilineans GyrA (XaA) forms a functional gyrase heterotetramer with E. coli GyrB (EcB) and can account for albicidin and quinolone resistance and low levels of relaxation activity. XaB probably contributes to both coumarin resistance and the distributive supercoiling pattern. Although XaB shows fewer apparent changes relative to EcB, the EcA.XaB hybrid relaxed DNA in the presence or absence of ATP and was unable to supercoil. A fuller understanding of structural differences between albicidin-sensitive and -resistant gyrases may provide new clues into features of the enzyme amenable to interference by novel antibiotics.
Assuntos
Antibacterianos , DNA Girase , Farmacorresistência Bacteriana Múltipla , Xanthomonas/efeitos dos fármacos , Xanthomonas/enzimologia , Sequência de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Clonagem Molecular , Biologia Computacional , DNA Girase/química , DNA Girase/efeitos dos fármacos , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Girase/metabolismo , DNA Super-Helicoidal/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Compostos Orgânicos/metabolismo , Compostos Orgânicos/farmacologia , Análise de Sequência de DNA , Xanthomonas/classificação , Xanthomonas/metabolismoRESUMO
The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Inibidores da Topoisomerase II , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/isolamento & purificação , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cumarínicos/química , Cumarínicos/farmacologia , DNA Girase/efeitos dos fármacos , DNA Girase/isolamento & purificação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Novobiocina/química , Novobiocina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genéticaRESUMO
Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.
Assuntos
Anti-Infecciosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium leprae/enzimologia , Quinolonas/farmacologia , Inibidores da Topoisomerase II , DNA/metabolismo , DNA Girase/isolamento & purificação , DNA Super-Helicoidal/efeitos dos fármacosRESUMO
DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. While all type II DNA topoisomerases relax supercoiled DNA, DNA gyrase is the only enyzme that introduces negative supercoils into DNA at the expense of ATP hydrolysis. We present here a biophysical characterization of the nucleotide cycle of DNA gyrase from Bacillus subtilis, both in the absence and presence of DNA. B. subtilis DNA gyrase is highly homologous to its well-studied Escherichia coli counterpart, but exhibits unique mechanistic features. The active heterotetramer of B. subtilis DNA gyrase is formed by mixing the GyrA and GyrB subunits. GyrB undergoes nucleotide-induced dimerization and is an ATP-operated clamp. The intrinsic ATPase activity of gyrase is stimulated tenfold in the presence of plasmid DNA. However, in contrast to the E. coli homolog, the rate-limiting step in the nucleotide cycle of B. subtilis GyrB is ATP hydrolysis, not product dissociation or an associated conformational change. Furthermore, there is no cooperativity between the two DNA and ATP binding sites in B. subtilis DNA gyrase. Nevertheless, the enzyme is as efficient in negative supercoiling as the E. coli DNA gyrase. Our results provide evidence that the evolutionary goal of efficient DNA supercoiling can be realized by similar architecture, but differences in the underlying mechanism. The basic mechanistic features are conserved among DNA gyrases, but the kinetics of individual steps can vary significantly even between closely related enzymes. This suggests that each topoisomerase represents a different solution to the complex reaction sequence in DNA supercoiling.